As researchers gain an increased understanding of the vertebrate immune system, methods to harness and specifically employ the immune system in preventing and fighting of illnesses are constantly being sought. Because of the immune system's extraordinary versatility, this approach, in principle, offers the possibility to react on any substance of sufficient size. Numerous attempts have been made to establish vaccination and/or immunological treatment methods of cancer, bacterial and viral infections.
Vaccination methods commonly employ exposing the organism to be vaccinated to vaccine preparations of an antigenic substance in order to generate an immune response. Particularly, the antigenic substance (antigen) is used to initiate production of immunoglobulins and/or cytotoxic phagocytic cells capable of detecting the antigenic substance itself or parts thereof (epitope), thereby becoming rapidly recognizable to the immune system. An antigen that has such an epitope becomes rapidly recognizable and can be inactivated or destroyed, e.g. by uptake into T cells and subsequent disintegration or by destruction of the cells comprising the antigen.
A similar method of vaccination or treatment is to extract lymphocytes, particularly lymphocyte stem cells, from the organism to be treated, exposing the extracted cells to the antigen, thus inducing production of immunoglobulins capable of detecting the antigen, and then reintroducing the immunoglobulin-producing lymphocytes into the organism to be treated (ex vivo treatment).
There are two general types of immune responses that contribute to the eradication of microbial infections and tumors. These are referred to as innate (natural, non-specific) and adaptive (acquired, specific) immune responses. Recently, several studies have demonstrated innate and adaptive immunity are linked in a variety of ways. Many types of adaptive immune responses are not successful in the absence of the appropriate collaborative innate stimulus.
In human peripheral blood, there are two subsets of professional antigen presenting cells (APC) that are the central coordinators of adaptive T cell responses. These are plasmacytoid (also called natural Interferon producing cells) dendritic cells (pDC) and myeloid dendritic cells (mDC). Both of these dendritic cell (DC) subsets express members of the Toll-like Receptor (TLR) family of pattern recognition receptors, so-called for their ability to bind conserved structural components of various microbes. Binding of such structures by TLRs initiates a signal transduction pathway that activates the DC for antigen presentation. During viral infections, pDCs activate mDCs by producing type I interferons (IFN-alpha) in response to ssRNA detected by the TLR8 pathway. Myeloid DCs derived from monocytes stimulated with the cytokines GM-CSF and IL4 can produce IL12 in response to bacterial structures such as lipopolysaccharide (LPS) and dsRNA via TLR4 and TLR3, respectively. Neither pathway is believed to result in the production of type I interferons in mDCs.
An important discovery has been the definition of tumor associated antigens (TAA) recognized by human T lymphocytes. The identification and molecular characterization of TAA is widely believed to have provided the means to create cancer vaccines. Current efforts in the creation of such vaccines are based on nucleic acid mediated immunization techniques, i.e. insertion of one or more antigen coding sequences (e.g. a TAA encoding sequence) into suitable expression (host) vectors capable of causing expression of the antigen coding sequence directly within transfected cells.
Commonly employed host vectors are bacteria and viruses or bacterial and viral genomes, respectively. Recent studies have shown that a cellular or encapsulated vector is not always necessary for vaccine preparation. Immunization with “naked” plasmid DNA and/or with RNA (e.g. with the nucleic acid being devoid of any other structural components such as proteins, lipids, or carbohydrates) can elicit powerful cellular and antibody responses. Nucleic acid vaccines, also termed recombinant vaccines, are thus vaccines in which the genome of the host vector integrates a nucleic acid sequence coding for an immunogen (antigen).
Compared to cell-based vaccines or cell lysates, recombinant vaccines have multiple advantages, the most prominent is probably that they can focus the immune response against a single, specific antigen like a TAA, and thus limit the possibility of releasing an uncontrolled autoimmune aggression against hitherto unknown antigens being present in normal tissues and tumor cells.
Currently, vaccination and therapeutic success vary greatly for different ailments and even among patients treated for the same ailment. Furthermore, vaccination does not always have satisfactory duration of effect, but can wear off within weeks. These drawbacks hold true also for a number of other vaccination methods, which may involve administration of live or inactivated vaccines. In general, vaccines are not always able to generate an appropriate and effective immune response by themselves.
Certain substances, when administered simultaneously with a specific antigen, will enhance the immune response to that antigen. Such substances, known as adjuvants, are routinely included in inactivated or purified antigen vaccines. Examples of adjuvants in common use are aluminium salts, liposomes and immunostimulating complexes (ISCOMS), complete and incomplete Freund's adjuvant, muramyl di-peptide and cytokines like interleukin (IL) 2, IL-12 and interferon (IFN) gamma.
Yet, while some adjuvants are suited for combination with some antigens or vaccines, they may fail in other combinations, or they may be toxic themselves to vertebrates like humans, promote poor cell mediated immunity, they may be unstable or too expensive and/or cumbersome to prepare. Improved methods for vaccination and treatment of illnesses and ailments in vertebrates are therefore needed.
Under certain circumstances, it is advantageous to boost patient's immune system without subsequent vaccination of the patient against a particular antigen. For example, patients suffering from diabetes have weakened immune responses that make them prone to different ailments, e.g. pneumonia or cancer. Patients with weakened immune responses will benefit greatly from development of medicaments that strengthen patients' immune system and improve patients' immune responses.
Generation of specific cell types, e.g. for tissue transplant purposes, is frequently needed. Among the cell types that can be used are dendritic cells and recently discovered fibrocytes.
Dendritic cells are potent antigen presenting cells. They are also reported to act as stimulators of a mixed lymphocyte reaction, to migrate selectively through tissues, to take up, process and present antigens, and serve as passenger cells that elicit rejection of transplanted tissues. For a review see for example Hart D N J “Dendritic cells: unique leukocyte populations which control the primary immune response”, Blood, 1997, 90:3245-3287. The study of dendritic cells offers potential applications in any field where the correct recognition of antigens and generation of immune responses is desirable. Such fields are, for example, transplantation medicine, vaccination, therapy of cancer and other illnesses connected with antigen presentation, prevention and treatment of autoimmune diseases and the like, see for example Dallal R M and Lotze M T, “Dendritic cells and human cancer vaccines”, Current Opinion in Immunology, 2000, 12:583-588. However, understanding of dendritic cells and their differentiation is insufficient. It is desirable to have methods for producing dendritic cells.
Current protocols for the production of dendritic cells rely on their differentiation from peripheral blood mononuclear cells, bone marrow cells or other CD34+ cells by exposing such cells to multiple cytokine combinations including granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), tumor necrosis factor alpha (TNFα), tumor growth factor beta (TGFβ) and IL-4. For a review see for example Strunk, D. et al., “Generation of human dendritic cells/Langerhans cells from circulating CD34+ hematopoietic progenitor cells”, Blood, 1996, 87:1292-1302 and Soligo, D. et al., “Expansion of dendritic cells derived from human CD34+ cells in static and continuous perfusion cultures”, British Journal of Hematology, 1998, 101:352-363. Likewise, dendritic cells have been produced from CD14+ blood monocytes and different maturation stages have been described. For a review see Winzler, C. et al., “Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures”, Journal of Experimental Medicine, 1997, 185:317-328 and U.S. Pat. No. 6,194,204 to Crawford and Chester. Yet, present protocols rely on expensive and unstable cytokine media components. Likewise, the yield of present protocols for the production of dendritic cells is often considered unsatisfactorily low and improvement in this area is needed.
Fibrocytes are a recently described type of cell characterized by their distinct phenotype (collagen+, CD34+), which normally is also vimetin+, CD13+ and CD45+. They are reported to enter rapidly from blood into subcutaneously implanted wound chambers. They are also frequently present in connective tissue scars and may play an important role in wound repair and pathological fibrotic responses. For a review see for example Bucala, R. et al., “Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair”, Mol. Med. 1994, 1:71-81, and Chesney J and Bucala R, “Peripheral blood fibrocytes: novel fibroblast-like cells that present antigen and mediate tissue repair”, Biochemical Society Transactions, 1997, 25:520-4. Like dendritic cells, protocols for the production of fibrocytes rely on expensive and unstable cytokine media components and provide often unsatisfactorily low yields and improvements in this area are likewise needed.
Serum amyloid P component (SAP) is a member of the pentraxin family of proteins (Bharadwaj et al., J. Immunology, 2001 166: 6735-6741). These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. CRP and SAP bind to leukocytes through Fc receptors for IgG (FcgammaR) (Bharadwaj et al., J. Immunology, 2001 166: 6735-6741).
Fc receptors (FcRs) are membrane receptors expressed on a number of immune effector cells. Upon interaction with target immunoglobulins, FcRs mediate a number of cellular responses, including, activation of cell mediated killing, induction of mediator release from the cell, uptake and destruction of antibody coated particles, and transport of immunoglobulins. Deo et al., 1997, Immunology Today 18:127-135. Further, it has been shown that antigen-presenting cells, e.g., macrophages and dendritic cells, undergo FcR mediated internalization of antigen-antibody complexes, allowing for antigen presentation and the consequent amplification of the immune response. As such, FcRs play a central role in development of antibody specificity and effector cell function. Deo et al., 1997, Immunology Today 18:127-135.
Each member of the Fc receptor family is defined by its specificity for a particular immunoglobulin isotype; Fc receptors for IgG antibodies are referred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Three subclasses of human gamma receptors have been identified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each human FcγR subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in Fcγ isoforms exists. The three genes encoding the human FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J Lab. Clin. Med. 126:330-41 (1995).
Because of the central role of FcγR as a trigger molecule in numerous immune responses, it has become a target for developing therapeutics. For example, several ongoing clinical trials are based on activating a cancer patient's effector cells by treating the patient with tumor-specific monoclonal antibodies (Mabs). These studies have shown that the tumor-specific antibodies mediate their effects in part through FcγR binding, and subsequent effector cell activity. (Adams et al., 1984, Proc. Natl. Acad. Sci. 81:3506-3510; Takahashi et al., 1995, Gastroenterology 108:172-182; Riethmeuller et al., 1994, Lancet 343:1177-1183, Clynes, R. A., Towers, T. L., Presta, L. G., and Ravetch, J. V., 2000, Nature Med. 6:443-446). Further, a novel series of bispecific molecule antibodies (BSMs), molecules engineered to have one arm specific for a tumor cell and the other arm specific for a target FcγR, are in clinical trials to specifically target a tumor for FcγR mediated, effector cell destruction of the tumor cells. (Valone et al., 1995, J. Clin. Oncol. 13:2281-2292; Repp et al., 1995, Hematother 4:415-421). FcγRs can also be used as therapeutic targets in infectious diseases and autoimmune disorders (Deo et al., 1997, Immunology Today 18:127-135; Ierino et al., 1993, J. Exp. Med. 178:1617-1628; Debre et al., 1993, Lancet 342:945-949). There is currently an unmet need for non-toxic modulators of Fc R.
If one or more of the above problems or needs could be addressed, a significant advance in the art would result.